Device for automatically counting blood cells



Sept. 24, 1957 J. c. PARKER ET A1. 2,807,416

DEVICE FOR AUTOMATICALLY COUNTING BLOOD CELLS Original Filed July 13,1955 2 Sheets-Sheet l wljmo mmm mut/ DOQ /Iv INVENTOR. JAMES c. PARKERBY wlLLsAM R. HORST d7- MM ATTORNEYS Sept. 24, 1957 J. c. PARKER ETAL807316 DEVICE FOR AUTOMATICALLY coUNTING BLOOD-CELLS original Filed July13, 1955 z'shets-sheet 2 FIG-5 r @5 IL' l INVENTOR.

JAMES C. PARKER WILLIAM R. HORST M 5 ATTORNEYS DEVICE FR AUT OMATICALLYCOUNTING BLOD CELLS James C. Parker and William R. Horst, Dayton, hio,as-

signors to The Commonwealth Engineering Company of Ghio, Dayton, Ohio, acorporation of Sirio Original application July 13, 1953, Serial No.367,708. Divided and this application November 26, 1954, Serial No.478,466

3 Claims. (Cl. 23S- 92) This invention relates to the counting ofparticles in physiological fluids and is particularly concerned with thecounting of blood cells. This application is a division of applicationSerial No. 367,708, liled Iuly 13, 1953.

Blood cell counts and knowledge of the numbers of the distinguishabletypes of blood cells are of value in the diagnosis of diseases forexample.

Counting methods currently in use involve in the determination, forexample, of the number of red and white blood cells in a given sample,the making up of two volumes; in one volume the red cells are countedand in the second the white cells are counted.

To effect the counting each sample is'diluted, appropriately stained andplaced in a counting chamber termed a hernocytometer and the number ofcells in a small iield `are counted under a microscope by a skilledtechnician.

It is diiticult with such a method to determine the exact theoreticalvolumes, the exact geometry of the counting chamber, and it is furthersubstantially impossible to 4secure an exactly representativedistribution of cells in the portion of the field under the microscope.

The errors are great particularly due to the fact that only a very smallportion of the total number of cells in an original sample volume areactually counted, for example, 500 in the case of a normal density of5,000,000 red cells per cubic millimeter. Surveys indicate that theerror probably is about il6 percent for a sample with 5,000,000 redcells per cubic millimeter and 122 per- -cent for a sample of 7,000white cells per cubic millimeter. ln many instances personal errors ofthe technician contribute to further deviation.

lt is a primary object of this invention to describe a new method forobtaining cell counts, which method eliminates errors due to imperfectdilution, counting chamber geometry, and non-representative viewingfields.

It is a further obejct of this invention to describe a novel method forcell counts which enables substantially all of the cells of the sampleto be counted.

It is an important object of this invention to describe a process whichpermits the simultaneous counting of blood cells of different types.

It is also an object of this invention to describe an apparatusarrangement which permits of quick accurate determination of the cellcounts.

The invention contemplates that the cells to be counted will be capableof absorbing light of one color while transmitting that of anotherc-olor. The cells are presented successively to a light which comprisesboth the color to be absorbed and the color to be transmitted.Absorption of the one color causes a decrease in intensity of that coloras well as of the total light, and this drop in intensity of the coloris used to actuate an electronic circuit arrangement including acounting unit. Such electronic circuit arrangements are in themselveswell known to the art and form no part of the present invention andaccordingly will be discussed hereinafter only in `suflicient detail toillustrate the invention.

In practice samples of blood, the red and white cells of nited tatsarent fice which .are to be counted, are lirst suspended in an appro- 1priate solution such as an isotonic solution, in order to form a dilutesuspension of the cells; the term dilute as used herein is intended toconvey the meaning that the concentration of the cells in the medium isconsiderably less than that occurring normally in the blood itself.

The cells are preferably stained in order to emphasize their colorabsorption properties; the staining of the cells under consideration isa Well known art and thus two types of cells in a suspension, as forexample the usual red and White cells, may be selectively stained in thepresence of each other. The stain may be formulated to enhance thenormal red coloration of the red cells and to simultaneously stain thewhite cells to a violet, indigo or blue. The particular stainformulation employed for a given operation may be readily selected andthe formulations in themselves form no part of this invention.

In thev method of invention then as practiced a dilute suspension ofstained red and white cells may be caused to fall by gravity in a narrowconduit which is itself provided with a constriction; this conduit andconstriction p and the dilution of the suspension permit the cells topass in substantially successive or sequential fashion through theconstriction and as they do so they are subjected to the light sourcereferred to hereinbefore.

The transmitted light decreased in intensity by the color absorption bythe cells is then directed through a filter onto a photocell which isresponsive to the decrease in the light intensity; the filter throughwhich this light passes in the counting of red blood cells for example,is a filter which normally would pass light of the color which isabsorbed by the red cells to be counted. Red cells, for example, absorbblue light and accordingly there will be a decrease in intensity of bluelight transmitted and a blue pass filter which prevents the passage ofcolors other than blue, if placed before the photocell, will permit theimpression thereon of blue light of varying intensity as the cells pass.A suitable electrical circuit actuated by the photocell notes thesechanges in intensity which each correspond to a cell passage andreflects the number of changes, that is the number of cells passing, ona counter.

The apparatus may be arranged to count the white cells at the same timethat the red cells are counted and all that is required for this purposeis that a lter also be provided which permits the passage of, forexample, red light if the white cells are stained violet. The photocellresponsive to the red light then actuates the counter in the mannerreferred to hereinbefore.

It is thus seen that it is only necessary to stain the cell distinctlyand to pass a portion of the light through the cells through variousfilters having the capacity for passing different light colors.

The invention will be more fully understood by reference to thefollowing detailed description and accompanying drawings wherein:

Figure l is a schematic view illustrating apparatus usable in thepractice of the method of invention;

Figure 2 is a view partially in section of the conduit and constrictionthrough which the cells pass to be counted;

Figure 3 shows a View on line 3--3 of Figure 2;

Figure 4 is a view partially in section illustrating slit means usefulin the practice of the invention in connection with the constriction;and

Figure 5 is a View illustrating another embodiment of the invention.

Referring to the drawings there is shown at 1 a vessel having asuspension medium 3 containing red blood cells 5 to be counted. Conduit7 which is preferably of small bore and only slightly larger than acapillary is connected to the support 9 for the orifice conduit 11,which conduit 11 as indicated at A is provided with a constriction.

As indicated in Figure l the suspension 3 and cells 5 when valve 6 isopen may flow under the influence of gravity through the conduits 7, 11and the constriction A to a receiver 23 wherein the cells are collectedas at 25. As noted hereinbefore the suspended medium is preferably :anisotonic solution such as 0.9 percentNaCl in distilled water.

The constriction A as shown more clearly in Figures 2 and 3 as well asthe conduit 11 is provided with the transparent support member 9 and achannel 10 exists for the passage of light through the member. Numerals13 and 15 indicate black masks which bound the light channel 10 andprevent the passage of light other than through the channel. Thus, asindicated at 17, the transparent region permits light to passtransversely as indicated by the arrows 19 and 21 through the member 9only at the area of the constriction A.

The. thickness or depth of theconstriction A, that is the dimensiontaken parallel to the light beam (Figure 3) should be sufiiciently smallto minimize the probability of simultaneous passage of a number of cellsin that dimension.` The width of the 'constriction may be as large as isconsistent with the generation of a detectable change in the total lightflux due to the passage of a single cell across a narrow slit (Figure 4)oriented in a plane perpendicular to the direction of flow ofthe cells.

It is to be particularly noted then that `the system is opaque exceptfor thatarea where the scanning slit 26 (Figure 4) and the transparentconstriction coincide which is indicated by the shaded `area in Figure4. n

For adequate resolution of the cells that may pass the slit almostsimultaneously in a horizontal line the slit 26 should have a dimensionconsiderably less than the diameter of a single cell. Since theproduction of an actual slit of such width is difiicult it is preferableto form an equivalent slit by optically magnifying the constriction orcounting orifice itself, which is effectively done by inu setting` amask 31 in the magnified real image of the constriction. This image isthen provided with a slit which may of course by considerably less widththan the slit at the constriction itself. A lens 29 inserted between themask'31 and the constriction facilitates the concentration of the lightflux passing through the constriction.

It is to be noted that with theabove arrangement the actual slit 32 ofthe mask 31 is considerably larger than the slit adjacenttheconstriction. The omission of the slit from the counting orifice orconstriction also removes the mask from destructive action of cleaningagents and permits manufacture on an economical basis.

As shownin Figure 1 for the counting of red blood cells a blue-passfilter 33 is passed between the mask 31 and photocell 35l which latteris connected to a differential network'37 and amplier V39 and counter`41. The elements 37,39 and 41 are standard equipment and accordinglyare not described iny detail. u

u In the operation ofthe device of Figure 1 the suspen sion togetherwith the small red blood cells moving through the constrictionA are4stained to absorb blue light;the cells pass before the light source 27and to the receiver 23. The light transmitted through the support 9 atthe constrictionApasses through the lens 29 to aperture 32 in the mask31 and then through the bluepass filter 33. u

Photocell35 is normallyin a conductive state under the influence` ofwhite lightpassing from the source 27 and since `only blue light willpass through the filter 33 it is blue light `which occasions the `normalfunctioning of photocell 35. As the cells pass through the constrictionthe intensity of blue light passing to .the filter 33 is` decreased andthis change of `energy passed to the photocell 35 pro-` In practice onlya small volume of suspension, for example a tenth of a cubic millimeter,isrequired in vessel 1 in order to attain an accurate counting of thecells. It is apparent however that a large volume of suspension 4and acorrespondingly large number of cells may be passed through theapparatus in a short time and that the counting arrangement is veryrapid. i

Figure 5 illustrates a further embodiment and the figure has identicalnumerals for the corresponding parts shown in Figure l. ln addition, inthe arrangement of Figure 5 there is shown at 43 a half-silvered mirrorwhich divides the light passing through the slit 32 of the mask 31 intotwo portions so that some of it strikes the bluepass filter 33 while theremainder is transmitted to the red-pass filter 45.

The elements 33, 35, 37, 39, 41 in this embodiment function precisely asthey do in connection withA the description of Figure l. However, in thepresent instance the suspension which is passed through the constrictioncontains both red and white cells, the red cells being as describedhereinbefore and the whitecells being stained to permit the absorptionof red light thereby.

Thus as the white cells pass through the white light source or thepolychromatic light source they absorb red light and the portion of thelight transmitted to the filter 45 will be decient in red light and thequantity of red light transmitted onto the photocell 47 will be reducedin intensity over that normally supplied. Accordingly a change occurs inthe output of the photocell 47 which is differentiated, amplified andcounted just as described in connection with Figure l. Accordingly meanshas been provided for the )counting of red and white cells at one time.Other similar cells could of course be counted in the same manner bysimply increasing thc number of filters and dispersing the lighttransmitted through the cells.

It is to be noted in connection with the foregoing that the electricalsignals are so differentiated by the network 37 and 39 that a signal istransmitted to the amplifier only when there is a change in photocellillumination. A steady electrical circuit .produced` by quiescentillumination of the photocell or slow drifts in illumination are nottransmitted to the amplifier. The amplifier is required to deliver asignal to operate the counter as at 41 or 53.

i With regard to the accuracy of the arrangement it is to be noted thatto insure of precision counting it is well to dilute the cells ratherhighly and by chemical treatment of the suspension to avoid theformation of rouleaux or clusters of cells. The dilution yof coursetends to avoid the presentation of more than one cell at a time to thescanning slit and constriction, whereby they might be counted as onecell which of course would tend to produce a low count. The clusters ofcells would have the same effect and should be avoided.

The dyes for the staining of the solutions are conventional as notedhereinbetore. It is not essential that the red cells be stained; butWrights solution may be employed for selective staining of the red andwhite cells for the accomplishment of the process of invention.Generally eosin in combination with methylene blue and other organicdyes is employed also for staining the cells.

It will be understood that this invention is susceptible to modificationin order to adopt it to different usages and conditions and accordingly,it is desired to compre hend such modifications within this invention asmay fall within the scope of the appended claims.

We claim:

p l. Apparatus for the counting of color-stained blood cells compri-singa support member having a conduit provided with a light transparentconstriction, said conduit and constriction being adapted for thepassage therethrough of a dilution liquid in which blood cells stainedin various colors are suspended for presenting the colorstained bloodcells successively to the constriction in a flow of the liquid past theconstriction, at least a pair of photo-electric responsive devicespositioned opposite the constriction and an automatic counting meansassociated with each of said devices, a source of white light positionedto direct light through the constriction of the conduit to thephoto-electric responsive devices, meansI responsive to the absorptionof a component of the white light by a colored-stained blood cellpassing the constriction to actuate one of the counting means, and meansresponsive to the absorption of another component of white light by aditerently stained blood cell to actuate another of the counting means.

2. Apparatus for the counting of color-stained blood cells comprising asupport member having a conduit provided with a light transparentconstriction, said conduit and constriction being adapted for thepassage therethrough of a dilution liquid in which blood cells stainedin various colors are suspended for presenting the colorstained bloodcells successively to the constriction in a liow of the liquid past theconstriction, at least a pair of photo-electric responsive devicespositioned opposite the constriction and an automatic counting meansassociated with each of said devices, a source of white light positionedto direct light through the constriction to the photo-electricresponsive devices, a iirst light filter `adapted to pass light of a rstcolor and to absorb others positioned between the constriction and afirst one of the photo-electric devices, a first one of thephoto-electric devices being normally responsive to light of said rstcolor emanating as a component of the White light and operable upon achange of intensity of the light of the rst color emanating from thewhite light source to actuate first counting means, a second light lteradapted to pass light of a second and diierent color and to absorb othercolors positioned between the constriction and the second of thephoto-electric devices to the side of the rst light iilter and operableupon a change of intensity of the light of the second color emanatingfrom the white light to actuate a second counting means, and means todirect white light passing through the constriction to the first andsecond lters simultaneously.

3. Apparatus for the counting of color-stained blood cells comprising asupport member having a conduit provided with a light transparentconstriction, said conduit and constriction being adapted for thepassage there through of a dilution liquid in which blood cells stainedin various colors are suspended for presenting the colorstained bloodcells successively to the constriction in a flow of the liquid past theconstriction, at least a pair of photo-electric responsive devicespositioned opposite the constriction and an automatic counting meansassociated with each of said devices, a source of White light posi--tioned to direct light through the constriction of the conduit to thephoto-electric responsive devices, a bluepass light iilter positionedbetween the constriction and a iirst one of the photo-electric devices,the rst one ofthe photo-electric devices being normally responsive toblue light emanating with said white light as a component of the whitelight of said source and operable upon a change of intensity of the bluelight passing the ilter to actuate a rst one of the counting means, ared-pass light lter positioned between the constriction and a second oneof the photofelectric devices and to one side of the blue-pass lter, thesecond of the photo-electric devices bein-g normally responsive to redlight emanating with said White light as a component of the white lightof said source and being operable upon a change of intensity of the redlight passing the lter to actuate a second of the counting means, andmeans to direct white light passing through the constriction to theblue-pass and the red-pass filters simultaneously.

References Cited in the le of this patent UNITED STATES PATENTS1,974,522 Turyman Sept. 25, 1934 2,369,577 Kielland Feb. 13, 19452,480,312 Wolf Aug. 30, 1949 2,584,082 Sandori et al Jan. 29, 19522,661,902 Wollt et al. Dec. 8, 1953

